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Novel approaches for disrupting gene expression in mammalian oocytes

破坏哺乳动物卵母细胞基因表达的新方法

基本信息

批准号：
8195724
负责人：
JANICE P EVANS
金额：
$ 24.6万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-24 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8195724
关键词：
Ablation Affinity Area Binding Biological Biology Cell Cycle Regulation Cell Nucleus Cells Clinical Trials Complex Contraceptive methods Cultured Cells Deoxyribonucleotides Development Disease Double-Stranded RNA Drug Delivery Systems Female Female Contraceptive Agents Fertility Fertilization Foundations Future Gene Delivery Gene Expression Gene Targeting Genetic Transcription Goals Human In Vitro Infertility Investigation Isomerism Knock-out Knockout Mice Knowledge Libraries Malignant neoplasm of prostate Mediating Meiotic Prophase I Messenger RNA Methodology Methods Microinjections Modification Mus Nucleic Acids Oligonucleotides Oocytes PLK1 gene Peptides Phenotype Process Proteins RNA RNA Interference Reagent Reproductive Health Research Research Methodology Research Proposals Residual state Small Interfering RNA Surface Testing Therapeutic Time Transcript Transfection Transgenic Organisms Vesicle Virus Diseases Work ZP1 ZP2 Zona Pellucida aptamer base cancer cell cell type ds-DNA genetic manipulation human disease in vitro testing in vivo insight interest mRNA Transcript Degradation novel novel strategies protein degradation tool zona pellucida glycoprotein

项目摘要

DESCRIPTION (provided by applicant): This R21 project seeks to develop new approaches for the genetic manipulation of mammalian oocytes, which we envision will accelerate advancing our knowledge of oocyte function and reproductive health. Genetic manipulation of mammalian oocytes has primarily used two methods: RNA interference and knockout mice. While RNAi has been a highly successful method of RNA ablation and subsequent protein knockdown in oocytes, RNAi approaches are not without limitations, as knockdown can be inefficient. Furthermore, double-stranded RNA and siRNAs have to be introduced into oocytes by microinjection, which is labor- and time-intensive and makes it impractical do use a large-scale RNAi approach (e.g., siRNA library-based screens). Knockout mice certainly have provided significant insights into mammalian oocyte biology as well, but knockout approaches also are not without pitfalls, including the time and expense involved in obtaining a knockout. This project seeks to develop alternatives to these methods, utilizing different established nucleic acid-based methods in novel combinations and with specialized modifications for the applications proposed here. In Aim 1, we will augment the use of siRNAs for post-transcriptional gene silencing with another reagent, a short single-stranded nucleic acid called a triplex-forming oligonucleotide (TFO) for pre-transcriptional silencing. TFOs bind to homopurine tracts in double-stranded DNA, and have been used to regulate gene expression in cultured cells and in vivo. The hypothesis for Aim 1 is that TFOs will inhibit transcription of a targeted gene, while siRNAs will mediate degradation of any residual mRNAs that were transcribed. This will be tested in vitro with isolated oocytes as well as with follicle-enclosed oocytes for longer-term culture. In Aim 2, we will develop methods for delivery of agents into oocytes. We will identify a novel agent for oocyte- specific delivery, using a screen of an aptamer library (with 1.2 X 1018 oligo-2'-deoxyribonucleotide sequence isomers) to isolate an aptamer that will interact with the oocyte's zona pellucida (ZP). Aptamers are nucleic acid-based molecules that bind with high affinity to target molecules. Aptamers can be used for delivery of agents such as siRNAs into cells; this delivery works in vivo, and aptamers currently are being developed as therapeutics to target drugs and other agents to specific cell types for treatment of a variety of diseases (13 aptamers are in clinical trials). Additionally, as an alternative tool, we will also test a cell-penetrating peptide for intra-oocyte delivery. We will couple siRNAs or TFOs to ZP-binding aptamers and/or a cell-penetrating peptide, and test these for their actions in oocytes. The future direction of this work will be to test the ZP-targeting aptamer for systemic delivery of siRNAs and TFOs, as a means of in vivo oocyte-specific knockdown as an alternative to knockout/transgenic methodologies, as well as potentially the foundation of a novel female contraceptive. PUBLIC HEALTH RELEVANCE: Investigations of the biology of the mammalian oocyte are of significant value, both for increasing our basic biological knowledge and for applications to human reproductive health. This project will accelerate the pace of research in this important area of reproductive health by developing new research methods for use in studying oocyte biology and female fertility and infertility, and for development of new female contraceptive methods.

描述（由申请人提供）：该R21项目旨在开发哺乳动物卵母细胞遗传操作的新方法，我们设想这将加速推进我们对卵母细胞功能和生殖健康的认识。哺乳动物卵母细胞的遗传操作主要使用两种方法：RNA干扰和敲除小鼠。虽然RNAi已经是在卵母细胞中进行RNA消融和随后的蛋白质敲低的非常成功的方法，但RNAi方法并非没有限制，因为敲低可能是低效的。此外，双链RNA和siRNA必须通过显微注射引入卵母细胞，这是劳动和时间密集型的，并且使得使用大规模RNAi方法（例如，基于siRNA文库的筛选）。敲除小鼠当然也为哺乳动物卵母细胞生物学提供了重要的见解，但敲除方法也并非没有陷阱，包括获得敲除所涉及的时间和费用。该项目旨在开发这些方法的替代方法，利用不同的已建立的基于核酸的方法进行新的组合，并针对本文提出的应用进行专门的修改。在目标1中，我们将增加使用siRNA进行转录后基因沉默，使用另一种试剂，一种称为三链体形成寡核苷酸（TFO）的短单链核酸进行转录前沉默。TF 0与双链DNA中的同型嘌呤段结合，并已用于调节培养细胞和体内的基因表达。目标1的假设是TFO将抑制靶基因的转录，而siRNA将介导转录的任何残留mRNA的降解。这将在体外用分离的卵母细胞以及卵泡封闭的卵母细胞进行长期培养。在目标2中，我们将开发用于将药剂递送到卵母细胞中的方法。我们将鉴定用于卵母细胞特异性递送的新型试剂，使用适体文库（具有1.2 X 1018寡-2 '-脱氧核糖核苷酸序列异构体）的筛选来分离将与卵母细胞的透明质酸（ZP）相互作用的适体。适体是以高亲和力结合靶分子的基于核酸的分子。适体可用于将诸如siRNA的试剂递送到细胞中;这种递送在体内起作用，并且适体目前正在被开发为将药物和其他试剂靶向特定细胞类型以治疗多种疾病的治疗剂（13种适体处于临床试验中）。此外，作为替代工具，我们还将测试用于卵母细胞内递送的细胞穿透肽。我们将siRNA或TFO与ZP结合适体和/或细胞穿透肽偶联，并测试它们在卵母细胞中的作用。这项工作的未来方向将是测试ZP靶向适体用于siRNA和TFO的全身递送，作为体内卵母细胞特异性敲除的手段，作为敲除/转基因方法的替代方法，以及潜在的新型女性避孕药的基础。公共卫生关系：哺乳动物卵母细胞的生物学研究具有重要价值，既可以增加我们的基本生物学知识，也可以应用于人类生殖健康。该项目将加快生殖健康这一重要领域的研究步伐，开发新的研究方法，用于研究卵母细胞生物学和女性生育力和不孕症，并开发新的女性避孕方法。